MATERNAL UNDERNOURISHMENT AND THE DEVELOPMENTAL PROGRAMMING OF ENERGY HOMEOSTASIS: A ROLE FOR THE GHRELIN RECEPTOR

Developmental programming in early life, conditioned by external cues, contributes to the susceptibility to develop metabolic disorders, particularly obesity and diabetes. Numerous epidemiological studies and animal studies have confirmed that maternal undernutrition and postnatal nutritional exposure can exert permanent and powerful effects on the programming of diseases in adulthood. Individuals are more likely to suffer from disease if a mismatch occurs between pre- and postnatal nutrient supply. The concept of the so-called "mismatch hypothesis" has been illustrated in the Dutch Famine study during the winter at the end of World War II, where famine exposure during pregnancy followed by sufficient food supply after birth resulted in an elevated rate of obesity or cardiovascular disease later in life in those exposed to famine. Hormonal and epigenetic factors are highly involved in early developmental programming of the offspring from maternal undernourished (UN) mothers. For example, the plasma leptin surge in the postnatal period is shifted and drastically reduced in UN rat offspring and disturbs the postnatal formation of hypothalamic neuronal circuits that are responsible for appropriate regulation of feeding and metabolism throughout life. The plasma leptin surge is followed by a surge of the hunger hormone ghrelin and recent studies have shown that postnatal ghrelin levels play a pivotal inhibitory role in limiting the excessive hypothalamic neuronal projections elicited by the leptin surge in normal nourished mice. However, it still unclear how maternal undernutrition affects plasma ghrelin levels during adequate lactation after birth and how ghrelin during early life is involved in the programming of long-term health. The aim of this PhD thesis was twofold. In the first aim, using a mouse "mismatch model" we investigated whether maternal undernutrition affected the magnitude and the timing in the increase in plasma ghrelin levels during adequate lactation after birth. Since ghrelin exerts its orexigenic role by activating GHSR in the arcuate nucleus of the hypothalamus to increase the release of AgRP and NPY, the effect on the development of AgRP fibers was investigated as well. The role of the GHSR was further investigated using maternal undernourished GHSR-/- mice and the effect on the metabolic disturbances in UN offspring on a high-fat diet (HFD) later in life was investigated. In the second aim, we investigated whether pharmacological treatment with the recently discovered endogenous GHSR antagonist LEAP2 (1-14) NH2 or the synthetic GHSR antagonist [D-Lys3]-GHRP-6 during lactation could reverse the obese phenotype of the offspring from undernourished mothers after feeding a HFD. Moreover, we investigated the impact of maternal undernutrition not only on central pathways but also on the plasticity of the ghrelin receptor involved in the regulation of neural contractions in the small intestine after treatment with GHSR antagonists. In the third chapter of this thesis, we developed a maternal undernourished mismatch model to investigate possible mechanisms in the programming of the thrifty phenotype. Smaller pups were born from UN mothers and a catch-up growth was observed in UN pups compared to NN pups during lactation with NN foster mothers. Plasma ghrelin levels showed a peak at postnatal day 15 (P15) which correlated with stomach ghrelin tissue content. This confirmed that the increase in plasma ghrelin levels was coming from the stomach. Maternal undernutrition did not affect the plasma ghrelin surge, nor did it affect age-dependent changes in Ghrelin and Goat mRNA expression in the stomach. In the hypothalamus, Ghsr and Agrp mRNA expression in UN pups were upregulated at P15 compared to NN pups. Previous studies have shown that ghrelin inhibited the density of AgRP fibers in the hypothalamus. In line with these findings and the upregulation of GHSR, the density of AgRP-IR fibers from the ARC to the PVN, one of the secondary order nuclei of ARC that are involved in food intake regulation, was significantly downregulated in UN pups compared to NN pups at P15. After weaning, body weight did not differ between NN and UN pups after 4 weeks of feeding with normal chow but significantly increased in UN offspring after 9 weeks on a HFD. Additionally, glucose tolerance was impaired in both groups but did not differ between NN and UN groups. This suggests that the hypothalamic rewiring during lactation may predispose the UN offspring to the increased body weight but that the HFD works as an additional trigger to increase body weight. To further investigated whether GHSR works as a key factor in this mismatch development, the experiment was repeated in wild type (WT) and GHSR-/- littermates using a similar mismatch model. In contrast to WT pups, maternal undernutrition failed to increase body weight of UN pups from GHSR-/- pups during lactation. After weaning, unlike the WT group, GHSR-/- UN offspring ate less food and gained less body weight after feeding with a HFD. The time-dependent changes after weaning in body weight between both maternal nutritional conditions were genotype-dependent. Body composition, measured at week 18 with Echo-MRI, showed that loss of lean mass rather than fat mass in GHSR-/- UN offspring contributed to the lower body weight. At week 15, metabolic measurements demonstrated that GHSR may have a protective role in the development of obesity by increasing oxygen consumption and ambulatory activity in UN offspring. Maternal undernutrition decreased heat production in both WT and GHSR-/- UN offspring compared to NN offspring during the night. This finding suggests that maternal undernutrition disturbed the thermogenesis of the offspring independent from GHSR. No genotype or group effect was observed in glucose intolerance. At week 19, no differences in the density of AgRP-IR fibers were observed between NN and UN offspring of both genotypes, indicating that the hypothalamic circuits were normalized at adulthood. In conclusion, studies in this chapter illustrated an important role of GHSR in the regulatory system of the energy balance that has been programmed for a thrifty postnatal lifestyle by maternal undernutrition but that has been challenged by adequate nutrition after birth. In the fourth chapter of this thesis, we investigated the effect of pharmacological targeting of the GHSR during the early postnatal period on reversing the phenotype of the maternal undernourished offspring that received adequate nutrition after birth. UN male pups were treated twice daily with saline, the endogenous ghrelin antagonist LEAP2 (1-14) NH2 or the synthetic antagonist D-Lys3-GHRP-6 from P5 to P15. NN male pups were treated with saline. Maternal undernutrition resulted in smaller pups at birth. In contrast to our previous study, UN pups failed to catch up growth compared to NN pups after fostering. The body weight gain retardation of UN pups during lactation might be due to the stress of the daily injections. Maternal undernourishment can be considered as a strong physiological stressor, because it is life threatening, UN pups might have been more vulnerable to the stress of the injections than the NN mice. Therefore, running non-injected NN and UN groups in parallel with saline injected NN and UN groups would have been more convincing to substantiate the stress response which was not anticipated. Probably as a result of this stress response, the upregulation of AgRP and GHSR was not observed anymore in the hypothalamus at P15 between saline NN and UN groups. GHSR antagonist treatment did not affect this hypothalamic expression nor did it change plasma ghrelin levels and ghrelin mRNA expression at P15 in UN groups. To investigate whether maternal undernutrition and the treatment with the GHSR antagonists during lactation affected peripheral GHSR activity in the myenteric plexus, neuronal contractions in jejunal segments were measured at P15. Isolated jejunal segments from saline treated UN pups, showed increased frequency-dependent excitatory responses compared to saline treated NN pups. This frequency-dependent neural hyperexcitability of the jejunal segments induced by maternal undernutrition was normalized by chronic treatment of UN pups with LEAP-2 (1-14) NH2 or [D-Lys3]-GHRP-6. After weaning, feeding with normal chow did not affect the body weight gain among groups. After HFD feeding, body weight gain of saline treated UN offspring remained lower compared to saline treated NN offspring. However, postnatal [D-Lys3]-GHRP-6 treatment but not LEAP-2 (1-14) NH2 treatment normalized the body weight of UN offspring. This indicated that postnatal GHSR blockage by [D-Lys3]-GHRP-6 could reverse the long-term effect of maternal undernutrition in a model where pre -and postnatal nutrient supply differed. No differences in food intake were observed among the different treated groups either on a normal diet or a HFD. Neither maternal undernourishment nor postnatal treatment with LEAP-2 (1-14) NH2 or [D-Lys3]-GHRP-6 affected plasma ghrelin levels and hypothalamic Ghsr and Agrp mRNA expression compared to saline treated NN offspring at week 17. Furthermore, postnatal [D-Lys3]-GHRP-6 treatment increased non-fasting plasma glucose levels in UN offspring compared to postnatal saline treated NN offspring at the time of sacrifice. These findings indicate that postnatal [D-Lys3]-GHRP-6 treatment did not only increase body weight gain but also induced hyperglycemia after HFD feeding. Because of the Covid 19 outbreak all experiments were aborted and oral glucose tolerance tests, metabolic measurements using metabolic cages and contractility studies could not be performed anymore during this PhD thesis. In conclusion, not only maternal undernutrition but also repeated injections during the postnatal period induced a stress response. Treatment of pups from undernourished mothers during the early postnatal period with [D-Lys3]-GHRP-6 but not with LEAP2 (1-14) NH2 could normalize the reduction in body weight gain induced by maternal undernutrition and postnatal stress after feeding with a HFD. Antagonism of GHSR could reduce the neural hyperexcitability of jejunal segments during lactation of maternal undernourished pups. To conclude, in this PhD thesis, our studies highlight an important role of GHSR in the early life developmental programming of energy homeostasis during maternal undernourishment and postnatal stress related responses in a mismatch model. Antagonism of the GHSR signaling pathway during early life might be a potential therapeutic strategy to prevent metabolic disorders in children born small for gestational age that live in an obesogenic environment.status: publishe